Dwindling fossil resources and concerns about greenhouse gas emissions are driving the development of alternative fuels (Hill, et. al. 2006). Cellulosic biofuels are among the leading alternative fuels because of their potential for high capacity, ability to be produced from non-food biomass, and relatively low feedstock cost (Klass, D. L., 2004). Today's cellulosic biofuels may have high production costs, particularly those associated with biomass pretreatment and enzymatic hydrolysis (Lynd, et. al., 2008; Himmel et. al., 2007). Enzyme loading requirements for cellulosic processing remain a challenge for the industry due to high costs and limited production capacity (Hood et. al., 2007). In contrast to microbial consolidated bioprocessing (Lynd, et. al., 2005), which relies on the availability of fermentable sugars for co-production of enzymes and biofuels, in planta consolidation is predicted to be more cost efficient because it does not require the diversion of fermentable sugars to microbial enzyme production (Sairam, et. al., 2008; Sainz 2009).
In spite of this advantage, in planta expression of cell wall degrading (CWD) enzymes may lead to detrimental plant phenotypes, including stunted plant stature, poor seed set and quality, reduced fertility, and increased susceptibility to disease (Harholt, et. al., 2010; Hood et. al., 2003; Taylor et. al., 2008); all of which can impact yield.
Inteins are self-splicing peptides found within host polypeptides (exteins) in many organisms (Perler et. al., 1994). Upon excision, inteins ligate the bordering extein polypeptide sequences back together with a peptide bond in a splicing reaction (Saleh and Perler, 2006). A cysteine, serine, or threonine at the junction site between the carboxy terminus of the intein and the carboxy extein of the target protein is often present (Xu, et. al., 1993).
Xylanases are a major class of cell wall degrading enzymes required for complete hydrolysis of plant cell walls into fermentable sugars. Xylanases hydrolyze hemicellulose polymers and play key roles in making cellulose more accessible to enzymatic hydrolysis (Selig et. al., 2008; Selig et. al., 2009; Dylan & Cann, 2009). Because of their catalytic properties, cellulases and xylanases that are able to function over a wide pH range and at high temperatures may be suitable in the production of biofuels and chemicals from lignocellulosic feedstocks. Process consolidation using in planta enzyme production has the potential to significantly reduce enzyme costs and production capacity, if it does not impact biomass yields.